Combination Therapy of Erythropoietin, Hydroxyurea, and Clotrimazole in a P Thalassemic Mouse: A Model for Human Therapy

Abstract

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.